91 research outputs found

    Diffusion MRI tractography for oncological neurosurgery planning:Clinical research prototype

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    Diffusion MRI tractography for oncological neurosurgery planning:Clinical research prototype

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    Surface-Based Computation of the Euler Characteristic in the BCC Grid

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    As opposed to the 3D cubic grid, the body-centered cubic (BCC) grid has some favorable topological properties: each set of voxels in the grid is a 3-manifold, with 2-manifold boundary. Thus, the Euler characteristic of an object O in this grid can be computed as half of the Euler characteristic of its boundary ∂O . We propose three new algorithms to compute the Euler characteristic in the BCC grid with this surface-based approach: one based on (critical point) Morse theory and two based on the discrete Gauss–Bonnet theorem. We provide a comparison between the three new algorithms and the classic approach based on counting the number of cells, either of the 3D object or of its 2D boundary surface

    Insights into the function of DNA repair factors MRN and ATM

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    DNA double strand breaks (DSB) are a particularly deleterious threat to genomic integrity throughout all domains of life. DSBs can cause chromosomal aberrations, tumorigenesis and cell death if left unre-paired and are caused by either endogenous or exogenous sources. Cells rely on efficient detection, repair and response upon occurrence of DSBs. In eukaryotes, DSBs are mostly repaired by either end joining pathways or homologous recombination (HR). HR, in contrast to the end joining pathways, en-ables error-free DSB repair in presence of a template sister chromatid. The Mre11-Rad50-Nbs1 (MRN) complex recognizes and tethers DNA ends, even if they are obstructed by proteins to initiate HR. In order to respond to DSBs, the MRN complex recruits and activates the signaling kinase Ataxia-telangiectasia mutated (ATM), that belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family. Activated ATM in turn initiates the cellular DNA damage response (DDR). Mre11 and Rad50 are highly conserved and form a topology-specific, ATP-dependent nuclease complex that pro-cesses DNA ends but leaves genomic DNA intact. The eukaryote specific Nbs1 subunit finetunes MRN’s endonuclease activity by providing interaction with proteins (e.g. CtIP). Apart from its nucleo-lytic activity, MRN has a scaffolding function that promotes DNA end tethering, repair foci formation and possibly signal amplification. Although the complex has been studied for more than two decades, a model that integrates both MRN’s enzymatic and scaffolding functions has not yet been established. In the first part of the thesis, such a model was elaborated by combining both structural and biochemical data from this and previ-ous studies. A cryo-electron microscopy (cryo-EM) structure of the Chaetomium thermophilum (Ct)MRN catalytic head domain in its ATPγS-bound state not only clarifies its atomic architecture but also reveals how a core part of Nbs1 stabilizes and possibly locks the Mre11 dimer. In this structure significant parts of the Rad50 coiled-coils were resolved in a rod configuration, stabilized by several interaction points. A previously uncharacterized C-terminal Mre11 domain, denoted bridge could fur-ther stabilize the rod configuration. The rod configuration and the bridge domain restrict access to the Rad50 DNA binding site. Biochemical analysis revealed the Rad50 DNA binding site is extremely specific for DNA ends. However, an additional, eukaryote-specific DNA binding site at the C-terminus of Mre11 enables binding to internal DNA. The Rad50 coiled-coil domains are linked at the apex via a zinc hook dimerization motif to form a large proteinaceous ring/rod. Cryo-EM data and crystal structures ex-plained how two MRN complexes can tether DNA ends via dimerization of these apical domains. In vivo assays indicate that mutation of the apex tethering element negatively impacts DSB repair. Mutations in DDR pathways allow cancer cells to cope with increased replication and genotoxic stress. For this reason, proteins involved in DDR were described to be promising targets in cancer therapy. Due to its central role in DSB induced DDR, ATM is an auspicious target for drug development. Howev-er, lack of ATM high-resolution structures, as well as atomic details of small molecule inhibitor binding modalities hampered the application of structure-based drug design. In the second part of the thesis, the binding modalities of two ATP-competitive ATM-inhibitors were described. This project was a col-laborative work with Merck KGaA, that provided a novel inhibitor (M4076) with improved pharmacoki-netics. Comparison of the inhibitor-bound kinase active sites with the likewise resolved ATPγS-bound active site explains the high affinities that were determined in biochemical assays. Superposition and sequence alignment of the ATM kinase active site with other PIKK active sites enables to rationalize the molecular reasons for selectivity. In biochemical assays, IC50 values of the inhibitors for ATM, PIKKs and CHK2 showed high selectivity towards ATM. The binding of the inhibitors stabilized the N-terminal solenoid domain of ATM, this enabled the generation of a high-resolution structure of the entire ATM protein. The quality of the map allowed the identification of two zinc binding sites that possibly stabi-lize loops and the generation of a near-complete ATM structure. Taken together, the structural data provides the framework for structure-based ATM inhibitor design and allows mapping of cancer muta-tion as well as functionally important protein interaction sites

    Holistic improvement of image acquisition and reconstruction in fluorescence microscopy

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    Recent developments in microscopic imaging led to a better understanding of intra- and intercellular metabolic processes and, for example, to visualize structural properties of viral pathogens. In this thesis, the imaging process of widefield and confocal scanning microscopy techniques is treated holistically to highlight general strategies and maximise their information content. Poisson or shot noise is assumed to be the fundamental noise process for the given measurements. A stable focus position is a basic condition for e.g. long-term measurements in order to provide reliable information about potential changes inside the Field-of-view. While newer microscopy systems can be equipped with hardware autofocus, this is not yet the widespread standard. For image-based focus analysis, different metrics for ideal, noisy and aberrated, in case of spherical aberration and astigmatism, measurements are presented. A stable focus position is also relevant in the example of 2-photon confocal imaging and at the same time the situation is aggravated in the given example, the measurement of the retina in the living mouse. In addition to the natural drift of the focal position, which can be evaluated by means of previously introduced metrics, rhythmic heartbeat, respiration, unrhythmic muscle twitching and movement of the mouse kept in artificial sleep are added. A dejittering algorithm is presented for the measurement data obtained under these circumstances. Using the additional information about the sample distribution in ISM, a method for reconstructing 3D from 2D image data is presented in the form of thick slice unmixing. This method can further be used for suppression of light generated outside the focal layer of 3D data stacks and is compared to selective layer multi-view deconvolution. To reduce phototoxicity and save valuable measurement time for a 3D stack, the method of zLEAP is presented, by which omitted Z-planes are subsequently calculated and inserted

    Advancing fluorescent contrast agent recovery methods for surgical guidance applications

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    Fluorescence-guided surgery (FGS) utilizes fluorescent contrast agents and specialized optical instruments to assist surgeons in intraoperatively identifying tissue-specific characteristics, such as perfusion, malignancy, and molecular function. In doing so, FGS represents a powerful surgical navigation tool for solving clinical challenges not easily addressed by other conventional imaging methods. With growing translational efforts, major hurdles within the FGS field include: insufficient tools for understanding contrast agent uptake behaviors, the inability to image tissue beyond a couple millimeters, and lastly, performance limitations of currently-approved contrast agents in accurately and rapidly labeling disease. The developments presented within this thesis aim to address such shortcomings. Current preclinical fluorescence imaging tools often sacrifice either 3D scale or spatial resolution. To address this gap in high-resolution, whole-body preclinical imaging tools available, the crux of this work lays on the development of a hyperspectral cryo-imaging system and image-processing techniques to accurately recapitulate high-resolution, 3D biodistributions in whole-animal experiments. Specifically, the goal is to correct each cryo-imaging dataset such that it becomes a useful reporter for whole-body biodistributions in relevant disease models. To investigate potential benefits of seeing deeper during FGS, we investigated short-wave infrared imaging (SWIR) for recovering fluorescence beyond the conventional top few millimeters. Through phantom, preclinical, and clinical SWIR imaging, we were able to 1) validate the capability of SWIR imaging with conventional NIR-I fluorophores, 2) demonstrate the translational benefits of SWIR-ICG angiography in a large animal model, and 3) detect micro-dose levels of an EGFR-targeted NIR-I probe during a Phase 0 clinical trial. Lastly, we evaluated contrast agent performances for FGS glioma resection and breast cancer margin assessment. To evaluate glioma-labeling performance of untargeted contrast agents, 3D agent biodistributions were compared voxel-by-voxel to gold-standard Gd-MRI and pathology slides. Finally, building on expertise in dual-probe ratiometric imaging at Dartmouth, a 10-pt clinical pilot study was carried out to assess the technique’s efficacy for rapid margin assessment. In summary, this thesis serves to advance FGS by introducing novel fluorescence imaging devices, techniques, and agents which overcome challenges in understanding whole-body agent biodistributions, recovering agent distributions at greater depths, and verifying agents’ performance for specific FGS applications

    Automated Deduction – CADE 28

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    This open access book constitutes the proceeding of the 28th International Conference on Automated Deduction, CADE 28, held virtually in July 2021. The 29 full papers and 7 system descriptions presented together with 2 invited papers were carefully reviewed and selected from 76 submissions. CADE is the major forum for the presentation of research in all aspects of automated deduction, including foundations, applications, implementations, and practical experience. The papers are organized in the following topics: Logical foundations; theory and principles; implementation and application; ATP and AI; and system descriptions

    Functional and structural substrates of increased dosage of Grik4 gene elucidated using multi-modal MRI

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    Grik4 is the gene responsible for encoding the high-affinity GluK4 subunit of the kainate receptors. Increased dosage of this subunit in the forebrain was linked to an increased level of anxiety, lack of social communication, and depression. On the synaptic level, abnormal synaptic transmission was also reported. The manifestations of this abnormal expression have not been investigated at the circuit level, nor the correlations between those circuits and the abnormal patterns of the behavior previously reported. In this line of work, we aspired to use different non-invasive magnetic resonance imaging (MRI) modalities to elucidate any disturbance that might stem from the increased dosage of Grik4 and how those changes might explain the abnormal behaviors. MRI offers a noninvasive way to look into the intact brain in vivo. Resting-state functional MRI casts light on how the brain function at rest on the network level and has the capability to detect any anomalies that might occur within or between those networks. On the microstructural level, the diffusion MRI is concerned with the underlying features of the tissues, using the diffusion of water molecules as a proxy for that end. Moving more macroscopically, using structural scans, voxel-based morphometry can detect subtle differences in the morphology of the different brain structures. We recorded videos of our animals performing two tasks that have long been linked to anxiety, the open field and the plus-maze tests before acquiring structural and functional scans. Lastly, we recorded blood-oxygenationlevel dependent (BOLD) signals in a different set of animals during electrical stimulation of specific white matter tracts in order to investigate how neuronal activity propagates. Our analysis showed a vast spectrum of changes in the transgenic group relative to the animals in the control group. On the resting-state networks level, we observed an increase in the within-network strength spanning different structures such as the hippocampus, some regions of the cortex, and the hypothalamus. The increased internal coherence or strength in the networks contrasted with a significant reduction in between-networks connectivity for some regions such as parts of the cortex and the hypothalamus, suggesting long-range network decorrelation. Supporting this idea, major white matter (WM) tracts, such as the corpus callosum and the hippocampal commissure, suffered from substantial changes compatible with an important reduction in myelination and/or a decrease in the mean axonal diameter. Macrostructurally speaking, the overexpression of GluK4 subunit had a bimodal effect, with expansion in some cortical areas in the transgenic animals accompanied by a shrinkage in the subcortical regions. Upon stimulating the brain with an electrical current, we noticed a difference in activity propagation between the two hemispheres. In transgenic animals, the evoked activity remained more confined to the stimulated hemisphere, again consistent with an impaired long-range connectivity. The structural changes both, at the micro and macro level, were in tight correlation with different aspects of the behavior including markers of anxiety such as the time spent in the open arms vs the closed arms in the plus-maze test and the time spent in the center vs the corners in the open field test. Our findings reveal how the disruption of kainate receptors, or more globally the glutamate receptors, and the abnormal synaptic transmission can translate into brain-wide changes in connectivity and alter the functional equilibrium between macro-and mesoscopic networks. The postsynaptic enhancement previously reported in the transgenic animals was here reflected in the BOLD signal and measured as an increase in the within-network strength. Importantly, the correlations between the structural changes and the behavior help to put the developmental changes and their behavioral ramifications into context. RESUMEN Grik4 es el gen responsable de codificar la subunidad GluK4 de alta afinidad de los receptores de kainato. El aumento de la dosis de esta subunidad en el prosencéfalo se relacionó con un mayor nivel de ansiedad, falta de comunicación social y depresión. A nivel sináptico, también se informó una transmisión sináptica anormal. Las manifestaciones de esta expresión anormal no se han investigado a nivel de circuito, ni las correlaciones entre esos circuitos y los patrones anormales de la conducta previamente informada. En esta línea de trabajo, aspiramos a utilizar diferentes modalidades de imágenes por resonancia magnética (MRI) no invasivas para dilucidar cualquier alteración que pudiera derivarse del aumento de la dosis de Grik4 y cómo esos cambios podrían explicar los comportamientos anormales. La resonancia magnética ofrece una forma no invasiva de observar el cerebro intacto in vivo. La resonancia magnética funcional en estado de reposo arroja luz sobre cómo funciona el cerebro en reposo en el nivel de la red y tiene la capacidad de detectar cualquier anomalía que pueda ocurrir dentro o entre esas redes. En el nivel microestructural, la resonancia magnética de difusión se ocupa de las características subyacentes de los tejidos utilizando la difusión de moléculas de agua como un proxy para ese fin. Moviéndose más macroscópicamente, utilizando escaneos estructurales, la morfometría basada en vóxeles puede detectar diferencias sutiles en la morfología de las diferentes estructuras cerebrales. Grabamos videos de nuestros animales realizando dos tareas que durante mucho tiempo se han relacionado con la ansiedad, el campo abierto y las pruebas de laberinto positivo antes de adquirir escaneos estructurales y funcionales. Por último, registramos señales dependientes del nivel de oxigenación de la sangre (BOLD) en un grupo diferente de animales durante la estimulación eléctrica de tractos específicos de materia blanca para investigar cómo se propaga la actividad neuronal. Nuestro análisis mostró un amplio espectro de cambios en el grupo transgénico en relación con los animales en el grupo de control. En el nivel de las redes de estado de reposo, observamos un aumento en la fuerza dentro de la red que abarca diferentes estructuras como el hipocampo, algunas regiones de la corteza y el hipotálamo. La mayor coherencia interna o fuerza en las redes contrastó con una reducción significativa en la conectividad entre redes para algunas regiones como partes de la corteza y el hipotálamo, lo que sugiere una descorrelación de redes de largo alcance. Apoyando esta idea, los grandes tractos de materia blanca (WM), como el cuerpo calloso y la comisura del hipocampo, sufrieron cambios sustanciales compatibles con una importante reducción de la mielinización y / o una disminución del diámetro axonal medio. Macroestructuralmente hablando, la sobreexpresión de la subunidad GluK4 tuvo un efecto bimodal, con expansión en algunas áreas corticales en los animales transgénicos acompañada de una contracción en las regiones subcorticales. Al estimular el cerebro con una corriente eléctrica, notamos una diferencia en la propagación de la actividad entre las dos hemiesferas. En los animales transgénicos, la actividad evocada permaneció más confinada al hemisferio estimulado, de nuevo consistente con una conectividad de largo alcance deteriorada. Los cambios estructurales, tanto a nivel micro como macro, estaban en estrecha correlación con diferentes aspectos de la conducta, incluidos marcadores de ansiedad como el tiempo pasado con los brazos abiertos frente a los brazos cerrados en la prueba del laberinto positivo y el tiempo pasado en el centro vs las esquinas en la prueba de campo abierto. Nuestros hallazgos revelan cómo la interrupción de los receptores de kainato, o más globalmente los receptores de glutamato, y la transmisión sináptica anormal pueden traducirse en cambios de conectividad en todo el cerebro y alterar el equilibrio funcional entre las redes macro y mesoscópicas. La mejora postsináptica informada anteriormente en los animales transgénicos se reflejó aquí en la señal BOLD y se midió como un aumento en la fuerza dentro de la red. Es importante destacar que las correlaciones entre los cambios estructurales y elcomportamiento ayudan a contextualizar los cambios en el desarrollo y sus ramificaciones conductuales
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